Anti-gm-csf antibodies and uses thereof

ABSTRACT

Provided are anti-GM-CSF antibodies or fragments thereof including humanized antibodies and fragments. Also provided are uses of the antibodies and fragments for therapeutic, diagnostic and prognostic purposes. Therapeutic uses of the antibodies and fragments, for example include the treatment of inflammatory and autoimmune diseases and disorders.

BACKGROUND

Granulocyte-macrophage colony-stimulating factor (GM-CSF or GM-CSF),also known as colony stimulating factor 2 (CSF2), is a monomericglycoprotein secreted by macrophages, T cells, mast cells, NK cells,endothelial cells and fibroblasts that functions as a cytokine. Thepharmaceutical analogs of naturally occurring GM-CSF are also referredto as sargramostim and molgramostim. Unlike granulocytecolony-stimulating factor, which specifically promotes neutrophilproliferation and maturation, GM-CSF affects more cell types, especiallymacrophages and eosinophils.

GM-CSF is a monomeric glycoprotein that functions as a cytokine. GM-CSFstimulates stem cells to produce granulocytes (neutrophils, eosinophils,and basophils) and monocytes. Monocytes exit the circulation and migrateinto tissue, whereupon they mature into macrophages and dendritic cells.Thus, it is part of the immune/inflammatory cascade, by which activationof a small number of macrophages can rapidly lead to an increase intheir numbers, a process crucial for fighting infection. GM-CSF also hassome effects on mature cells of the immune system. These include, forexample, inhibiting neutrophil migration and causing an alteration ofthe receptors expressed on the cells surface.

GM-CSF signals via signal transducer and activator of transcription,STAT5. In macrophages, it has also been shown to signal via STAT3. Thecytokine activates macrophages to inhibit fungal survival. It inducesdeprivation in intracellular free zinc and increases production ofreactive oxygen species that culminate in fungal zinc starvation andtoxicity. Thus, GM-CSF facilitates development of the immune system andpromotes defense against infections. GM-CSF also plays a role inembryonic development by functioning as an embryokine produced byreproductive tract.

GM-CSF is manufactured using recombinant DNA technology and is marketedas a protein therapeutic called molgramostim or, when the protein isexpressed in yeast cells, sargramostim. It is used as a medication tostimulate the production of white blood cells and thus preventneutropenia following chemotherapy. GM-CSF has also been evaluated inclinical trials for its potential as a vaccine adjuvant in HIV-infectedpatients.

Inhibition of GM-CSF, by contrast, can be useful for treating diseasessuch as inflammatory diseases and autoimmune disorders includingrheumatoid arthritis (OA), multiple sclerosis (MS) and plaque psoriasis.Inhibition of GM-CSF can also be useful for treating cancer.

SUMMARY

The present disclosure provides anti-GM-CSF antibody having high bindingaffinity to human GM-CSF proteins and having potent activitiesinhibiting the binding of GM-CSF to its receptor. These anti-GM-CSFantibodies are useful for therapeutic purposes such as treating varioustypes of inflammatory diseases, autoimmune disorders and cancers, andcan also be used for diagnostic and prognostic purposes.

The present disclosure, in one embodiment, provides an isolated antibodyor fragment thereof, wherein the antibody or fragment thereof hasspecificity to a human GM-CSF protein and comprises a VH CDR1 of SEQ IDNO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO:6. In some embodiments, the antibody or fragment thereof furthercomprises a heavy chain constant region, a light chain constant region,an Fc region, or the combination thereof. In some embodiments, the lightchain constant region is a kappa or lambda chain constant region. Insome embodiments, the antibody or fragment thereof is of an isotype ofIgG, IgM, IgA, IgE or IgD. In some embodiments, the isotype is IgG1,IgG2, IgG3 or IgG4. In some embodiments, the antibody or fragmentthereof is a chimeric antibody, a humanized antibody, or a fully humanantibody.

In some embodiments, the antibody or fragment thereof is a humanizedantibody. In some embodiments, the antibody or fragment thereofcomprises a heavy chain variable region comprising one or more aminoacid residues selected from the group consisting of: (a) Glu at position1, (b) Arg at position 98, (c) Ser at position 72, (d) Ala at position68, (e) Leu at position 70, Ile at position 48, (g) Asp at position 26,and (h) Leu at position 29, according to Kabat numbering, andcombinations thereof.

In some embodiments, the heavy chain variable region comprises afragment of DYTLT (SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) starting atposition 26 according to Kabat numbering.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising one or more amino acid residuesselected from the group consisting of: (a) Ala at position 46, (b) Aspat position 60, (c) Asp at position 70, (d) Ser at position 43, and (f)Phe at position 87, according to Kabat numbering, and combinationsthereof.

In some embodiments, the antibody or fragment thereof of comprises aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8-17, or a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 8-17. In some embodiments, the heavychain variable region comprises the amino acid sequence of SEQ ID NO:11, 14 or 17.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 19-22, or a peptide having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 19-22. In some embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 19 or22.

In some embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 14 and the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 22. In some embodiments,the antibody or fragment thereof is a bispecific antibody or singlechain variable fragment.

The present disclosure, in one embodiment, provides an isolated antibodyor fragment thereof, wherein the antibody or fragment thereof hasspecificity to a human GM-CSF protein and comprises a VH CDR1 of SEQ IDNO: 23, a VH CDR2 of SEQ ID NO: 24, a VH CDR3 of SEQ ID NO: 25, a VLCDR1 of SEQ ID NO: 26, a VL CDR2 of SEQ ID NO: 27, and a VL CDR3 of SEQID NO: 28. In some embodiments, the antibody or fragment thereof furthercomprises a heavy chain constant region, a light chain constant region,an Fc region, or the combination thereof. In some embodiments, the lightchain constant region is a kappa or lambda chain constant region. Insome embodiments, the antibody or fragment thereof is of an isotype ofIgG, IgM, IgA, IgE or IgD. In some embodiments, the isotype is IgG1,IgG2, IgG3 or IgG4. In some embodiments, the antibody or fragmentthereof is a chimeric antibody, a humanized antibody, or a fully humanantibody.

In some embodiments, the antibody or fragment thereof is a humanizedantibody. In some embodiments, the antibody or fragment thereofcomprises a heavy chain variable region comprising one or more aminoacid residues selected from the group consisting of E1, R84, Y27, 128,148, T68, L70, or T30, according to Kabat numbering, and combinationsthereof.

In some embodiments, the heavy chain variable region comprises afragment of GYIFT (SEQ ID NO: 44), GYIFS (SEQ ID NO: 45), or GGTFS (SEQID NO: 46) starting at position 26 according to Kabat numbering.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising one or more amino acid residuesselected from the group consisting of: V48, D57, Q70 or S43, accordingto Kabat numbering, and combinations thereof.

In some embodiments, the antibody or fragment thereof of comprises aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 29-35, or a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 29-35. In some embodiments, the heavychain variable region comprises the amino acid sequence of SEQ ID NO: 34or 35.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 36-41, or a peptide having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 36-41. In some embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 38 or39.

In some embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 34 and the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 38. In some embodiments,the heavy chain variable region comprises the amino acid sequence of SEQID NO: 35 and the light chain variable region comprises the amino acidsequence of SEQ ID NO: 39. In some embodiments, the antibody or fragmentthereof is a bispecific antibody or single chain variable fragment.

In one embodiment, provided is a composition comprising the antibody orfragment thereof of the present disclosure and a pharmaceuticallyacceptable carrier. Also provided, in one embodiment, is an isolatedcell comprising one or more polynucleotide encoding the antibody orfragment thereof of the present disclosure.

In one embodiment, the present disclosure provides a method of treatingan inflammatory or autoimmune disease or condition in a patient in needthereof, comprising administering to the patient the antibody orfragment thereof, or a composition of the present disclosure. Alsoprovided are uses of the antibody or fragment thereof, or a compositionfor the manufacture of a medicament for treating an inflammatory orautoimmune disease or condition.

In some embodiments, the inflammatory disease or condition is selectedfrom the group consisting of Alzheimer's disease, Addison's disease,atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA),rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosingspondylitis, asthma, atherosclerosis, chronic obstructive pulmonarydisease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis,fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupuserythematous (SLE), nephritis, Parkinson's disease (PD), vasculitis, andulcerative colitis.

Also provided, in one embodiment, is a method of reducing or relievingpain in a patient in need thereof, comprising administering to thepatient the antibody or fragment thereof of the present disclosure.

In some embodiments, the autoimmune disease or condition is selectedfrom the group consisting of alopecia areata, autoimmune hemolyticanemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), celiacdisease, autoimmune juvenile idiopathic arthritis, glomerulonephritis,Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenicpurpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis,pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,polymyositis, primary biliary cirrhosis, psoriasis, rheumatoidarthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemiclupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis,autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis(Wegener's).

Also provided is a method of detecting expression of GM-CSF in a sample,comprising contacting the sample with the antibody or fragment thereofunder conditions for the antibody or fragment thereof to bind to theGM-CSF, and detecting the binding which indicates expression of GM-CSFin the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results form confirmatory ELISA binding assays forselecting primary hybridoma clones for subcloning.

FIG. 2 shows results form confirmatory ELISA binding assays forselecting primary hybridoma clones for subcloning.

FIG. 3 shows dose-dependent binding of the test antibodies to humanGM-CSF.

FIG. 4 plots the binding kinetics of antibodies with recombinant humanGM-CSF.

FIG. 5 shows dose-dependent inhibition of GM-CSF binding to the GM-CSFreceptor alpha by the antibodies.

FIG. 6 shows that the antibodies significantly decreased the level ofpSTAT5 activation induced by GM-CSF.

FIG. 7 shows the inhibition of GM-CSF dependent TF-1 proliferation bythe antibodies.

FIG. 8 shows that all the humanized antibodies demonstrated potentbinding potency against human GM-CSF.

FIG. 9 shows that a few humanized antibodies exhibited strongestinhibition of TF-1 proliferation.

FIG. 10 shows that the tested antibodies effectively blocked pSTAT5signaling.

FIG. 11 shows dose-dependent binding of the antibodies to rhesus GM-CSF.

FIG. 12 plots the pharmacokinetics parameters of Hu23F4-27.

DETAILED DESCRIPTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

The term “isolated” as used herein with respect to cells, nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs orRNAs, respectively, that are present in the natural source of themacromolecule. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to cells or polypeptides which are isolated fromother cellular proteins or tissues. Isolated polypeptides is meant toencompass both purified and recombinant polypeptides.

As used herein, the term “recombinant” as it pertains to polypeptides orpolynucleotides intends a form of the polypeptide or polynucleotide thatdoes not exist naturally, a non-limiting example of which can be createdby combining polynucleotides or polypeptides that would not normallyoccur together.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalentpolynucleotides are those having the above-noted specified percenthomology and encoding a polypeptide having the same or similarbiological activity.

The term “an equivalent nucleic acid or polynucleotide” refers to anucleic acid having a nucleotide sequence having a certain degree ofhomology, or sequence identity, with the nucleotide sequence of thenucleic acid or complement thereof. A homolog of a double strandednucleic acid is intended to include nucleic acids having a nucleotidesequence which has a certain degree of homology with or with thecomplement thereof. In one aspect, homologs of nucleic acids are capableof hybridizing to the nucleic acid or complement thereof. Likewise, “anequivalent polypeptide” refers to a polypeptide having a certain degreeof homology, or sequence identity, with the amino acid sequence of areference polypeptide. In some aspects, the sequence identity is atleast about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects,the equivalent polypeptide or polynucleotide has one, two, three, fouror five addition, deletion, substitution and their combinations thereofas compared to the reference polypeptide or polynucleotide. In someaspects, the equivalent sequence retains the activity (e.g.,epitope-binding) or structure (e.g., salt-bridge) of the referencesequence.

Hybridization reactions can be performed under conditions of different“stringency”. In general, a low stringency hybridization reaction iscarried out at about 40° C. in about 10×SSC or a solution of equivalentionic strength/temperature. A moderate stringency hybridization istypically performed at about 50° C. in about 6×SSC, and a highstringency hybridization reaction is generally performed at about 60° C.in about 1×SSC. Hybridization reactions can also be performed under“physiological conditions” which is well known to one of skill in theart. A non-limiting example of a physiological condition is thetemperature, ionic strength, pH and concentration of Mg²⁺ normally foundin a cell.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching. The term “polymorphism” refers to the coexistence of morethan one form of a gene or portion thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene”. Apolymorphic region can be a single nucleotide, the identity of whichdiffers in different alleles.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA,miRNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this disclosure that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, an “antibody” or “antigen-binding polypeptide” refers toa polypeptide or a polypeptide complex that specifically recognizes andbinds to an antigen. An antibody can be a whole antibody and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule having biological activityof binding to the antigen. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein.

The terms “antibody fragment” or “antigen-binding fragment”, as usedherein, is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, scFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the intact antibody.The term “antibody fragment” includes aptamers, spiegelmers, anddiabodies. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion proteinof the variable regions of the heavy (V_(H)) and light chains (V_(L)) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the VH with the C-terminus ofthe VL, or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. ScFv molecules are known in the art and aredescribed, e.g., in U.S. Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides thatcan be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as gamma, mu, alpha, delta,or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g.,γ1-γ4). It is the nature of this chain that determines the “class” ofthe antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgG₅,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are clearly within the scope ofthe present disclosure, the following discussion will generally bedirected to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Antibodies, antigen-binding polypeptides, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aVK or VH domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto LIGHT antibodies disclosed herein). Immunoglobulin or antibodymolecules of the disclosure can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VK) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CK) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CK domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VK domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VK chains (i.e.CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances,e.g., certain immunoglobulin molecules derived from camelid species orengineered based on camelid immunoglobulins, a complete immunoglobulinmolecule may consist of heavy chains only, with no light chains. See,e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen-binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen-binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen-binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen-binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference in their entireties. The CDR definitions according to Kabatand Chothia include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or variants thereof isintended to be within the scope of the term as defined and used herein.The appropriate amino acid residues which encompass the CDRs as definedby each of the above cited references are set forth in the table belowas a comparison. The exact residue numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which residues comprise aparticular CDR given the variable region amino acid sequence of theantibody.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3  95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to table above, the Kabat number system describes the CDRregions as follows: CDR-H1 begins at approximately amino acid 31 (i.e.,approximately 9 residues after the first cysteine residue), includesapproximately 5-7 amino acids, and ends at the next tryptophan residue.CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includesapproximately 16-19 amino acids, and ends at the next arginine or lysineresidue. CDR-H3 begins at approximately the thirty third amino acidresidue after the end of CDR-H2; includes 3-25 amino acids; and ends atthe sequence W-G-X-G, where X is any amino acid. CDR-L1 begins atapproximately residue 24 (i.e., following a cysteine residue); includesapproximately 10-17 residues; and ends at the next tryptophan residue.CDR-L2 begins at approximately the sixteenth residue after the end ofCDR-L1 and includes approximately 7 residues. CDR-L3 begins atapproximately the thirty third residue after the end of CDR-L2 (i.e.,following a cysteine residue); includes approximately 7-11 residues andends at the sequence F or W-G-X-G, where X is any amino acid.

Antibodies disclosed herein may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, donkey,rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, an antigen-binding polypeptide for use in the disclosuremay comprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In another embodiment, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, anantibody for use in the disclosure may lack at least a portion of a CH2domain (e.g., all or part of a CH2 domain). As set forth above, it willbe understood by one of ordinary skill in the art that the heavy chainconstant region may be modified such that they vary in amino acidsequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG₁ molecule and a hinge region derived from an IgG₃ molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG₁ molecule and, in part, from anIgG₃ molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG₁ molecule and, in part,from an IgG₄ molecule.

As used herein, the term “light chain constant region” includes aminoacid sequences derived from antibody light chain. Preferably, the lightchain constant region comprises at least one of a constant kappa domainor constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983). The CH2 domain is unique inthat it is not closely paired with another domain. Rather, two N-linkedbranched carbohydrate chains are interposed between the two CH2 domainsof an intact native IgG molecule. It is also well documented that theCH3 domain extends from the CH2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J. Immunol161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CK regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant disclosure)is obtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, “percent humanization” is calculated by determining thenumber of framework amino acid differences (i.e., non-CDR difference)between the humanized domain and the germline domain, subtracting thatnumber from the total number of amino acids, and then dividing that bythe total number of amino acids and multiplying by 100.

By “specifically binds” or “has specificity to,” it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope D.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of anautoimmune disease. Beneficial or desired clinical results include, butare not limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sport, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “to a patient in need of treatment” or“a subject in need of treatment” includes subjects, such as mammaliansubjects, that would benefit from administration of an antibody orcomposition of the present disclosure used, e.g., for detection, for adiagnostic procedure and/or for treatment.

Anti-GM-CSF Antibodies

The present disclosure provides anti-GM-CSF antibodies with highaffinity to the human GM-CSF protein. The tested antibodies exhibitedpotent binding and inhibitory activities and are useful for therapeuticand diagnostics uses. In addition to the original murine antibodies, thehumanized ones also showed strong binding affinity to rhesus GM-CSF andhuman GM-CSF, which binding blocked the GM-CSF's binding to GM-CSFreceptor alpha and blocked GM-CSF induced pSTAT5 signaling, andinhibited GM-CSF dependent TF-1 proliferation.

In accordance with one embodiment of the present disclosure, provided isan antibody that includes the heavy chain and light chain variabledomains with the CDR regions as defined in SEQ ID NO: 1-6 or SEQ ID NO:23-28, as shown below.

TABLE 1a Sequences of the CDR regions of 23F4 Name Sequences SEQ ID NO:VH CDR1 SHYLH 1 VH CDR2 WIFPGDDKTKYNEKFKG 2 VH CDR3 GTKYLNWNFDV 3VL CDR1 KANQNVGTTLA 4 VL CDR2 SASYRYS 5 VL CDR3 HQYTTYPLT 6

TABLE 1b Sequences of the CDR regions of 50C5 Name Sequences SEQ ID NO:VH CDR1 PYSIH 23 VH CDR2 YINPSTGYIEYNQHFKD 24 VH CDR3 GGDYEGYFDY 25VL CDR1 RLNENIYSFLA 26 VL CDR2 NAETLAE 27 VL CDR3 QQHYGTPYT 28

As demonstrated in the experimental examples, the antibodies thatcontained these CDR regions, whether mouse, humanized or chimeric, hadpotent GM-CSF binding and inhibitory activities. In some embodiments, ananti-GM-CSF antibody of the present disclosure includes the VH and VLCDR as listed in Table 1a-b, with one, two or three furthermodifications. Such modifications can be addition, deletion orsubstitution of amino acids.

In some embodiments, the modification is substitution at no more thanone residues from each of the CDRs. In some embodiments, themodification is substitution at one, two or three residues. In oneembodiment, the modification is substitution at one of the residues.Such substitutions, in some embodiments, are conservative substitutions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide is preferably replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Non-limiting examples of conservative amino acid substitutions areprovided in the table below, where a similarity score of 0 or higherindicates conservative substitution between the two amino acids.

TABLE 2 Amino Acid Similarity Matrix C G P S A T D E N Q H K R V M I L FY W W −8 −7 −6 −2 −6 −5 −7 −7 −4 −5 −3 −3 2 −6 −4 −5 −2 0 0 17 Y 0 −5 −5−3 −3 −3 −4 −4 −2 −4 0 −4 −5 −2 −2 −1 −1 7 10 F −4 −5 −5 −3 −4 −3 −6 −5−4 −5 −2 −5 −4 −1 0 1 2 9 L −6 −4 −3 −3 −2 −2 −4 −3 −3 −2 −2 −3 −3 2 4 26 I −2 −3 −2 −1 −1 0 −2 −2 −2 −2 −2 −2 −2 4 2 5 M −5 −3 −2 −2 −1 −1 −3−2 0 −1 −2 0 0 2 6 V −2 −1 −1 −1 0 0 −2 −2 −2 −2 −2 −2 −2 4 R −4 −3 0 0−2 −1 −1 −1 0 1 2 3 6 K −5 −2 −1 0 −1 0 0 0 1 1 0 5 H −3 −2 0 −1 −1 −1 11 2 3 6 Q −5 −1 0 −1 0 −1 2 2 1 4 N −4 0 −1 1 0 0 2 1 2 E −5 0 −1 0 0 03 4 D −5 1 −1 0 0 0 4 T −2 0 0 1 1 3 A −2 1 1 1 2 S 0 1 1 1 P −3 −1 6 G−3 5 C 12

TABLE 3 Conservative Amino Acid Substitutions For Amino AcidSubstitution With Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys ArginineD-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-GluGln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-GlnCysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser GlutamineD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-AlaIsoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val,Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-OrnMethionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-ProSerine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His,D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

In some embodiments, an antibody or fragment thereof includes no morethan one, no more than two, or no more than three of the abovesubstitutions.

In some embodiments, the antibody or fragment thereof has specificity toa human GM-CSF protein and comprises a VH CDR1 of SEQ ID NO: 1, a VHCDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO:4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.Non-limiting examples of VH are provided in SEQ ID NO: 7-17, out ofwhich SEQ ID NO: 7 is the mouse VH, and SEQ ID NO: 8-17 are humanizedones. Further, these humanized VH include one or more back-mutations tothe mouse version. Likewise, non-limiting examples of VL (VK) areprovided in SEQ ID NO: 18-22. SEQ ID NO: 18 is a mouse sequence, and SEQID NO: 19-22 are humanized sequences, among which SEQ ID NO: 20-22include one or more back-mutations, as shown in the examples.

The back-mutations are shown to be useful for retaining certaincharacteristics of the anti-GM-CSF antibodies. Accordingly, in someembodiments, the anti-GM-CSF antibodies of the present disclosure, inparticular the human or humanized ones, include one or more of theback-mutations. In some embodiments, the VH back-mutation (i.e.,included amino acid at the specified position) is one or more selectedfrom (a) Glu at position 1 (E1), (b) Arg at position 98 (R98), (c) Serat position 72 (S72), (d) Ala at position 68 (A68), (e) Leu at position70 (L70), (f) Ile at position 48 (148), (g) Asp at position 26 (D26),and (h) Leu at position 29 (L29), according to Kabat numbering, andcombinations thereof.

In some embodiments, the humanized antibody includes at least VHback-mutation E1. In some embodiments, the humanized antibody includesat least VH back-mutations E1 and R98. In some embodiments, thehumanized antibody includes at least VH back-mutations E1 and another aslisted above. In some embodiments, the humanized antibody includes atleast VH back-mutation group (E1, R98 and S72), (E1, R98, S72 and A68),(E1, R98, S72, A68, L70 and 148), (E1, R98, S72, A68, L70, 148, D26 andL29), (E1 and S72), (E1, S72 and L70), (E1, S72, L70, 148 and A68), (E1,S72, L70, 148, A68, D26 and L29).

In some embodiments, the heavy chain variable region comprises afragment of DYTLT (SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) at theN-terminal end of the CDR1, i.e., starting at position 26 according toKabat numbering. In one embodiment, the heavy chain variable regioncomprises DYTLT (SEQ ID NO: 42). In one embodiment, the heavy chainvariable region comprises GYTFT (SEQ ID NO: 43).

In some embodiments, the humanized antibody includes one or more of theback-mutations. In some embodiments, the VL back-mutation is one or moreselected from (a) Ala at position 46 (A46), (b) Asp at position 60(D60), (c) Asp at position 70 (D70), (d) Ser at position 43 (S43), and(0 Phe at position 87 (F87), according to Kabat numbering, andcombinations thereof.

In some embodiments, the humanized antibody includes at least two, threeor four of VL back-mutations A46, D60, D70, S43, or F87. In someembodiments, the humanized antibody includes at least VL back-mutationA46. In some embodiments, the humanized antibody includes at least VLback-mutations A46 and D60 and another as listed above. In someembodiments, the humanized antibody includes at least VL back-mutationgroup (A46, D60 and D70) or (A46, D60, D70, S43 and F87).

In some embodiments, the humanized antibody includes at least VHback-mutations (E1, R98, S72, A68, L70 and 148) and no VLback-mutations. In some embodiments, the humanized antibody includes atleast VH back-mutations (E1, S72, L70, 148, A68, D26 and L29) and no VLback-mutations. In some embodiments, the humanized antibody includes atleast VH back-mutations (E1 and S72) and VL back-mutations (A46, D60,D70, S43 and F87).

In some embodiments, the anti-GM-CSF antibody of the present disclosureincludes a VH of SEQ ID NO: 8-17, and a VL of SEQ ID NO: 19-22, or theirrespective biological equivalents. A biological equivalent of a VH or VLis a sequence that includes the designated amino acids while having anoverall 80%, 85%, 90%, 95%, 98% or 99% sequence identity. A biologicalequivalent of SEQ ID NO: 10, therefore, can be a VH that has an overall80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 10 butretains the CDRs (SEQ ID NO: 1-3 or their variants), and optionallyretains one or more, or all of the back-mutations.

In one embodiment, the VH has the amino acid sequence of SEQ ID NO: 11and the VL has the amino acid sequence of SEQ ID NO: 19. In oneembodiment, the VH has the amino acid sequence of SEQ ID NO: 17 and theVL has the amino acid sequence of SEQ ID NO: 19. In one embodiment, theVH has the amino acid sequence of SEQ ID NO: 11 and the VL has the aminoacid sequence of SEQ ID NO: 22. Each of the recited sequences, it isnoted, can also be substituted with their biological equivalents.

In some embodiments, the antibody or fragment thereof has specificity toa human GM-CSF protein and comprises a VH CDR1 of SEQ ID NO: 23, a VHCDR2 of SEQ ID NO: 24, a VH CDR3 of SEQ ID NO: 25, a VL CDR1 of SEQ IDNO: 26, a VL CDR2 of SEQ ID NO: 27, and a VL CDR3 of SEQ ID NO: 28.Non-limiting examples of VH are provided in SEQ ID NO: 29-35, out ofwhich SEQ ID NO: 29 is the mouse VH, and SEQ ID NO: 30-35 are humanizedones. Further, these humanized VH include one or more back-mutations tothe mouse version. Likewise, non-limiting examples of VL (VK) areprovided in SEQ ID NO: 36-41. SEQ ID NO: 36 is a mouse sequence, and SEQID NO: 37-41 are humanized sequences, among which SEQ ID NO: 38-41include one or more back-mutations, as shown in the examples.

The back-mutations are shown to be useful for retaining certaincharacteristics of the anti-GM-CSF antibodies. Accordingly, in someembodiments, the anti-GM-CSF antibodies of the present disclosure, inparticular the human or humanized ones, include one or more of theback-mutations. In some embodiments, the VH back-mutation (i.e.,included amino acid at the specified position) is one or more selectedfrom E1, R84, Y27, 128, 148, T68, L70, or T30, according to Kabatnumbering, and combinations thereof.

In some embodiments, the humanized antibody includes at least VHback-mutation E1. In some embodiments, the humanized antibody includesat least VH back-mutations E1 and R84. In some embodiments, thehumanized antibody includes at least VH back-mutations E1 and another aslisted above. In some embodiments, the humanized antibody includes atleast VH back-mutation group (E1), (E1 and R84), (E1, R84, Y27 and 128),(E1, R84, Y27, 128 and 148), (E1, R84, Y27, 128, 148, T68 and L70), or(E1, R84, Y27, 128, 148, T68, L70 and T30).

In some embodiments, the heavy chain variable region comprises afragment of GYIFT (SEQ ID NO: 44), GYIFS (SEQ ID NO: 45), or GGTFS (SEQID NO: 46) at the N-terminal end of the CDR1, i.e., starting at position26 according to Kabat numbering. In one embodiment, the heavy chainvariable region comprises GYIFT (SEQ ID NO: 44). In one embodiment, theheavy chain variable region comprises GYIFS (SEQ ID NO: 45). In oneembodiment, the heavy chain variable region comprises GGTFS (SEQ ID NO:46).

In some embodiments, the humanized antibody includes one or more of theback-mutations. In some embodiments, the VL back-mutation is one or moreselected from V48, D57, Q70 or S43, according to Kabat numbering, andcombinations thereof.

In some embodiments, the humanized antibody includes at least two, threeor four of VL back-mutations V48, D57, Q70 or S43. In some embodiments,the humanized antibody includes at least VL back-mutation V48. In someembodiments, the humanized antibody includes at least VL back-mutationsV48 and D57 and another as listed above. In some embodiments, thehumanized antibody includes at least VL back-mutation group (V48), (V48and D57), (V48, D57 and Q70) or (V48, D57, Q70 and S43).

In some embodiments, the humanized antibody includes at least VHback-mutations (E1, R84, Y27, 128, 148, T68 and L70) and VLback-mutation V48. In some embodiments, the humanized antibody includesat least VH back-mutations (E1, R84, Y27, 128, 148, T68, L70 and T30)and VL back-mutations (V48 and D57).

In some embodiments, the anti-GM-CSF antibody of the present disclosureincludes a VH of SEQ ID NO: 30-35, and a VL of SEQ ID NO: 37-41, ortheir respective biological equivalents. A biological equivalent of a VHor VL is a sequence that includes the designated amino acids whilehaving an overall 80%, 85%, 90%, 95%, 98% or 99% sequence identity. Abiological equivalent of SEQ ID NO: 35, therefore, can be a VH that hasan overall 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ IDNO: 35 but retains the CDRs (SEQ ID NO: 23-25 or their variants), andoptionally retains one or more, or all of the back-mutations.

In one embodiment, the VH has the amino acid sequence of SEQ ID NO: 34and the VL has the amino acid sequence of SEQ ID NO: 38. In oneembodiment, the VH has the amino acid sequence of SEQ ID NO: 35 and theVL has the amino acid sequence of SEQ ID NO: 39. Each of the recitedsequences, it is noted, can also be substituted with their biologicalequivalents.

It will also be understood by one of ordinary skill in the art thatantibodies as disclosed herein may be modified such that they vary inamino acid sequence from the naturally occurring binding polypeptidefrom which they were derived. For example, a polypeptide or amino acidsequence derived from a designated protein may be similar, e.g., have acertain percent identity to the starting sequence, e.g., it may be 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the startingsequence.

The antibodies and fragments of the present disclosure can bemono-specific or bispecific antibodies or fragments, in someembodiments. For a bispecific antibody, the other specificity can be toa different target epitope of GM-CSF or a different target protein whichis useful for a particular use, e.g., therapeutic use. In one aspect,the target protein is s cytokine such as TNF-alpha, IL-6, IL-1, andIL-17. In another aspect, the target protein is a chemokine, such asCCL2, CXCL12, and CXCL13. In another aspect, the target protein is acell surface protein, such as CD3, CSF-1R, CD20, and CD73.

In certain embodiments, the antibody comprises an amino acid sequence orone or more moieties not normally associated with an antibody. Exemplarymodifications are described in more detail below. For example, anantibody of the disclosure may comprise a flexible linker sequence, ormay be modified to add a functional moiety (e.g., PEG, a drug, a toxin,or a label).

Antibodies, variants, or derivatives thereof of the disclosure includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment does notprevent the antibody from binding to the epitope. For example, but notby way of limitation, the antibodies can be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the antibodies may contain one or more non-classicalamino acids.

In some embodiments, the antibodies may be conjugated or connected byother means to another molecule to form a bi-functional molecule. Thesecond molecule may be one of therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG. Some non-limiting examples are cytokinesor other soluble factors, such as IL-10, IL-25, IL-27, IL-33, IL-35, andIL-36. Also provided, in some embodiments, are antibody-drug conjugateswhich include an antibody or fragment of the present disclosure and asmall molecule drug.

The antibodies may be conjugated or fused to a therapeutic agent, whichmay include detectable labels such as radioactive labels, animmunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivetherapeutic or diagnostic agent, a cytotoxic agent, which may be a drugor a toxin, an ultrasound enhancing agent, a non-radioactive label, acombination thereof and other such agents known in the art.

The antibodies can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantigen-binding polypeptide is then determined by detecting the presenceof luminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

The antibodies can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). Techniques for conjugatingvarious moieties to an antibody are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.,(eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. (52:119-58 (1982)).

Polynucleotides Encoding the Antibodies and Methods of Preparing theAntibodies

The present disclosure also provides isolated polynucleotides or nucleicacid molecules encoding the antibodies, variants or derivatives thereofof the disclosure. The polynucleotides of the present disclosure mayencode the entire heavy and light chain variable regions of theantigen-binding polypeptides, variants or derivatives thereof on thesame polynucleotide molecule or on separate polynucleotide molecules.Additionally, the polynucleotides of the present disclosure may encodeportions of the heavy and light chain variable regions of theantigen-binding polypeptides, variants or derivatives thereof on thesame polynucleotide molecule or on separate polynucleotide molecules.

Methods of making antibodies are well known in the art and describedherein. In certain embodiments, both the variable and constant regionsof the antigen-binding polypeptides of the present disclosure are fullyhuman. Fully human antibodies can be made using techniques described inthe art and as described herein. For example, fully human antibodiesagainst a specific antigen can be prepared by administering the antigento a transgenic animal which has been modified to produce suchantibodies in response to antigenic challenge, but whose endogenous locihave been disabled. Exemplary techniques that can be used to make suchantibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592;6,420,140 which are incorporated by reference in their entireties.

In certain embodiments, the prepared antibodies will not elicit adeleterious immune response in the animal to be treated, e.g., in ahuman. In one embodiment, antigen-binding polypeptides, variants, orderivatives thereof of the disclosure are modified to reduce theirimmunogenicity using art-recognized techniques. For example, antibodiescan be humanized, primatized, deimmunized, or chimeric antibodies can bemade. These types of antibodies are derived from a non-human antibody,typically a murine or primate antibody, that retains or substantiallyretains the antigen-binding properties of the parent antibody, but whichis less immunogenic in humans. This may be achieved by various methods,including (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies; (b) grafting at leasta part of one or more of the non-human complementarity determiningregions (CDRs) into a human framework and constant regions with orwithout retention of critical framework residues; or (c) transplantingthe entire non-human variable domains, but “cloaking” them with ahuman-like section by replacement of surface residues. Such methods aredisclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498(1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are herebyincorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T-cell epitopes (see, e.g., InternationalApplication Publication Nos.: WO/9852976 A1 and WO/0034317 A2). Forexample, variable heavy chain and variable light chain sequences fromthe starting antibody are analyzed and a human T-cell epitope “map” fromeach V region showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence is created. Individual T-cell epitopes from the T-cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative variable heavy and variable lightsequences are designed comprising combinations of amino acidsubstitutions and these sequences are subsequently incorporated into arange of binding polypeptides. Typically, between 12 and 24 variantantibodies are generated and tested for binding and/or function.Complete heavy and light chain genes comprising modified variable andhuman constant regions are then cloned into expression vectors and thesubsequent plasmids introduced into cell lines for the production ofwhole antibody. The antibodies are then compared in appropriatebiochemical and biological assays, and the optimal variant isidentified.

The binding specificity of antigen-binding polypeptides of the presentdisclosure can be determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

Alternatively, techniques described for the production of single-chainunits (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Hustonet al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al.,Nature 334:544-554 (1989)) can be adapted to produce single-chain unitsof the present disclosure. Single-chain units are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single-chain fusion peptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242: 1038-1041 (1988)).

Examples of techniques which can be used to produce single-chain Fvs(scFvs) and antibodies include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88(1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerraet al., Science 240:1038-1040 (1988). For some uses, including in vivouse of antibodies in humans and in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See, e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J.Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816397, which are incorporated herein by reference intheir entireties.

Humanized antibodies are antibody molecules derived from a non-humanspecies antibody that bind the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Often,framework residues in the human framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen-binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen-binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973(1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which isincorporated by reference in its entirety).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can alsobe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 72:899-903(1988). See also, U.S. Pat. No. 5,565,332, which is incorporated byreference in its entirety.)

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

Additionally, using routine recombinant DNA techniques, one or more ofthe CDRs of the antigen-binding polypeptides of the present disclosure,may be inserted within framework regions, e.g., into human frameworkregions to humanize a non-human antibody. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds to at least one epitopeof a desired polypeptide, e.g., LIGHT. Preferably, one or more aminoacid substitutions may be made within the framework regions, and,preferably, the amino acid substitutions improve binding of the antibodyto its antigen. Additionally, such methods may be used to make aminoacid substitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentdisclosure and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA:851-855 (1984);Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule, ofappropriate antigen specificity, together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in thedisclosure as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Additionally, standard techniques known to those of skill in the art canbe used to introduce mutations in the nucleotide sequence encoding anantibody of the present disclosure, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference variable heavy chainregion, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1,CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomlyalong all or part of the coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity.

Treatment Methods and Uses

As described herein, the antibodies, variants or derivatives of thepresent disclosure may be used in certain treatment and diagnosticmethods.

The present disclosure is further directed to antibody-based therapieswhich involve administering the antibodies of the disclosure to apatient such as an animal, a mammal, and a human for treating one ormore of the disorders or conditions described herein. Therapeuticcompounds of the disclosure include, but are not limited to, antibodiesof the disclosure (including variants and derivatives thereof asdescribed herein) and nucleic acids or polynucleotides encodingantibodies of the disclosure (including variants and derivatives thereofas described herein).

The antibodies and fragments of the present disclosure, in someembodiments, can be used for manufacture of a medicament for treating aninflammatory or autoimmune disease or disorder, or cancer. It is alsobelieved that the antibodies and fragments of the present disclosure areuseful for treating the underlying mechanisms of pain or the painitself.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was originallyknown by its ability to generate colonies of both granulocytes andmacrophages from bone marrow precursors. It has also been shown to acton mature myeloid cells as pro-survival, activation, and differentiationfactors. Recent studies suggest that GM-CSF also has manypro-inflammatory functions and plays critical roles in the developmentof autoimmune and inflammatory diseases.

GM-CSF promotes the survival and activation of macrophages, neutrophils,and eosinophils, as well as dendritic cell (DC) maturation. GM-CSF canpolarize macrophages into M1-like inflammatory macrophages, whichproduce a variety of inflammatory cytokines such as TNF, IL-6, IL-12p70,IL-23, or IL-1β, and thus promote Th1-Th17 responses. On the other hand,the association of GM-CSF and Th2 immunity is also reported in allergicairway inflammation.

GM-CSF receptor consists of an α-subunit which binds GM-CSF with lowaffinity (GMRα) and a signal-transducing βc-subunit which is shared withthe IL-3 and IL-5 receptors. The binary complex of GM-CSF and GMRαinteracts with a free βc-subunit and forms the high-affinity hexamercomplex. Dodecamer complexes formed by lateral aggregation of twohexamer complexes enable Jak2 associated with a βc-subunit to dimerizeand transphosphorylate, but the hexamer complexes do not. This structureleads to dose-dependent responses of GM-CSF receptor activation. Lowconcentration of GM-CSF, as in normal condition, causes βc Ser585phosphorylation and activates 14-3-3/PI-3 kinase pathway which onlyleads to cell survival. Higher concentration of GM-CSF, as ininflammatory condition, turns off βc Ser585 phosphorylation and mediatedβc Tyr577 phosphorylation and activation of Jak2/STAT5 pathway,Ras/mitogen-activated protein kinase pathway, and PI-3 kinase pathway,resulting in promotion of cell survival, proliferation, and activation.

A wide variety of cells can produce GM-CSF. Major sources of GM-CSF areT and B cells, monocyte/macrophage endothelial cells, and fibroblasts.Neutrophils, eosinophils, epithelial cells, mesothelial cells, Panethcells, chondrocytes, and tumor cells can also produce GM-CSF. Theproduction of GM-CSF is stimulated by various factors, including TNF,IL-1, toll-like receptor agonists, and prostaglandin E2. Recently, thepathogenicity of GM-CSF-producing CD4 T cells in autoimmune andinflammatory diseases is clarified and gaining increasing attention.

Recent evidence revealed that GM-CSF played critical roles in thedevelopment of many autoimmune diseases. GM-CSF depletion orneutralization suppresses many autoimmune disease models, includingexperimental autoimmune encephalomyelitis (EAE), arthritis,arthritis-related interstitial lung disease, nephritis, or psoriasis. Itis also suggested that inhibition of GM-CSF can be useful for treatingcancer.

In some embodiments, the inflammatory disease or condition to be treatedby the disclosed antibodies, fragments and compositions includes one ormore of Alzheimer's disease, Addison's disease, atherosclerosis,ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoidarthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis,asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD),Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia,hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous(SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerativecolitis.

In some embodiments, the autoimmune disease or condition to be treatedby the disclosed antibodies, fragments and compositions includes one ormore of alopecia areata, autoimmune hemolytic anemia, autoimmunehepatitis, dermatomyositis, diabetes (type 1), celiac disease,autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves'disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura,myasthenia gravis, autoimmune myocarditis, multiple sclerosis,pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,polymyositis, primary biliary cirrhosis, psoriasis, rheumatoidarthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemiclupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis,autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis(Wegener's).

Rheumatoid arthritis (RA) is a long-term autoimmune disorder thatprimarily affects joints. It typically results in warm, swollen, andpainful joints. Pain and stiffness often worsen following rest. Mostcommonly, the wrist and hands are involved, with the same jointstypically involved on both sides of the body. The disease may alsoaffect other parts of the body. While the cause of rheumatoid arthritisis not clear, it is believed to involve a combination of genetic andenvironmental factors. The underlying mechanism involves the body'simmune system attacking the joints. This results in inflammation andthickening of the joint capsule. The goals of treatment are to reducepain, decrease inflammation, and improve a person's overall functioning.Pain medications, steroids, and NSAIDs are frequently used to help withsymptoms. A group of medications called disease-modifying antirheumaticdrugs (DMARDs), such as hydroxychloroquine and methotrexate, may be usedto try to slow the progression of disease.

Osteoarthritis (OA) is a type of joint disease that results frombreakdown of joint cartilage and underlying bone. The most commonsymptoms are joint pain and stiffness. Initially, symptoms may occuronly following exercise, but over time may become constant. Othersymptoms may include joint swelling, decreased range of motion, and whenthe back is affected weakness or numbness of the arms and legs. Causesinclude previous joint injury, abnormal joint or limb development, andinherited factors. Risk is greater in those who are overweight, have oneleg of a different length, and have jobs that result in high levels ofjoint stress. Osteoarthritis is believed to be caused by mechanicalstress on the joint and low grade inflammatory processes. Treatmentincludes exercise, efforts to decrease joint stress, support groups, andpain medications.

Multiple sclerosis (MS) is a demyelinating disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. This damage disrupts the ability of parts of the nervous systemto communicate, resulting in a range of signs and symptoms, includingphysical, mental, and sometimes psychiatric problems. Specific symptomscan include double vision, blindness in one eye, muscle weakness,trouble with sensation, or trouble with coordination. While the cause isnot clear, the underlying mechanism is thought to be either destructionby the immune system or failure of the myelin-producing cells. There isno known cure for multiple sclerosis. Treatments attempt to improvefunction after an attack and prevent new attacks.

Asthma is a common long-term inflammatory disease of the airways of thelungs. It is characterized by variable and recurring symptoms,reversible airflow obstruction, and bronchospasm. Symptoms includeepisodes of wheezing, coughing, chest tightness, and shortness ofbreath. Asthma is thought to be caused by a combination of genetic andenvironmental factors. Environmental factors include exposure to airpollution and allergens. Asthma is classified according to the frequencyof symptoms, forced expiratory volume in one second (FEV1), and peakexpiratory flow rate. It may also be classified as atopic or non-atopic,where atopy refers to a predisposition toward developing a type 1hypersensitivity reaction. There is no cure for asthma. Symptoms can beprevented by avoiding triggers, such as allergens and irritants, and bythe use of inhaled corticosteroids. Long-acting beta agonists (LABA) orantileukotriene agents may be used in addition to inhaledcorticosteroids if asthma symptoms remain uncontrolled. Treatment ofrapidly worsening symptoms is usually with an inhaled short-actingbeta-2 agonist such as salbutamol and corticosteroids taken by mouth. Invery severe cases, intravenous corticosteroids, magnesium sulfate, andhospitalization may be required.

Chronic obstructive pulmonary disease (COPD) is a type of obstructivelung disease characterized by long-term poor airflow. COPD can includetwo main conditions, emphysema and chronic bronchitis. In emphysema, thewalls between many of the air sacs are damaged. As a result, the airsacs lose their shape and become floppy. This damage also can destroythe walls of the air sacs, leading to fewer and larger air sacs insteadof many tiny ones. If this happens, the amount of gas exchange in thelungs is reduced. In chronic bronchitis, the lining of the airways staysconstantly irritated and inflamed, and this causes the lining to swell.Lots of thick mucus forms in the airways, making it hard to breathe.There is no known cure for COPD, but the symptoms are treatable and itsprogression can be delayed.

Pain is a distressing feeling often caused by intense or damagingstimuli, such as stubbing a toe, burning a finger, putting alcohol on acut, or bumping the “funny bone”. Pain is a complex, subjectivephenomenon, defining pain has been a challenge. Pain is also referred toas an unpleasant sensory and emotional experience associated with actualor potential tissue damage. Pain is sometimes regarded as a symptom ofan underlying condition, such as inflammation.

In some embodiments, provided are methods for treating a cancer in apatient in need thereof. The method, in one embodiment, entailsadministering to the patient an effective amount of an antibody of thepresent disclosure.

Non-limiting examples of cancers include bladder cancer, breast cancer,colorectal cancer, endometrial cancer, esophageal cancer, head and neckcancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma,melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

Additional diseases or conditions associated with increased cellsurvival, that may be treated, prevented, diagnosed and/or prognosedwith the antibodies or variants, or derivatives thereof of thedisclosure include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular antibodies,variant or derivative thereof used, the patient's age, body weight,general health, sex, and diet, and the time of administration, rate ofexcretion, drug combination, and the severity of the particular diseasebeing treated. Judgment of such factors by medical caregivers is withinthe ordinary skill in the art. The amount will also depend on theindividual patient to be treated, the route of administration, the typeof formulation, the characteristics of the compound used, the severityof the disease, and the desired effect. The amount used can bedetermined by pharmacological and pharmacokinetic principles well knownin the art.

Methods of administration of the antibodies, variants or include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The antigen-bindingpolypeptides or compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Thus, pharmaceutical compositions containingthe antigen-binding polypeptides of the disclosure may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may bedesirable to introduce the antibodies of the disclosure into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

It may be desirable to administer the antigen-binding polypeptides orcompositions of the disclosure locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., inconjunction, with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an antibody, of the disclosure,care must be taken to use materials to which the protein does notabsorb.

In another embodiment, the antigen-binding polypeptide or compositioncan be delivered in a vesicle, in particular a liposome (see Langer,1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapyof Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327;see generally ibid.)

In yet another embodiment, the antigen-binding polypeptide orcomposition can be delivered in a controlled release system. In oneembodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,N. Engl. J Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the therapeutic target, i.e., thebrain, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

In a specific embodiment where the composition of the disclosurecomprises a nucleic acid or polynucleotide encoding a protein, thenucleic acid can be administered in vivo to promote expression of itsencoded protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The amount of the antibodies of the disclosure which will be effectivein the treatment, inhibition and prevention of an inflammatory, immuneor malignant disease, disorder or condition can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease, disorder orcondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

As a general proposition, the dosage administered to a patient of theantigen-binding polypeptides of the present disclosure is typically 0.1mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of thedisclosure may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The methods for treating an infectious or malignant disease, conditionor disorder comprising administration of an antibody, variant, orderivative thereof of the disclosure are typically tested in vitro, andthen in vivo in an acceptable animal model, for the desired therapeuticor prophylactic activity, prior to use in humans. Suitable animalmodels, including transgenic animals, are well known to those ofordinary skill in the art. For example, in vitro assays to demonstratethe therapeutic utility of antigen-binding polypeptide described hereininclude the effect of an antigen-binding polypeptide on a cell line or apatient tissue sample. The effect of the antigen-binding polypeptide onthe cell line and/or tissue sample can be determined utilizingtechniques known to those of skill in the art, such as the assaysdisclosed elsewhere herein. In accordance with the disclosure, in vitroassays which can be used to determine whether administration of aspecific antigen-binding polypeptide is indicated, include in vitro cellculture assays in which a patient tissue sample is grown in culture, andexposed to or otherwise administered a compound, and the effect of suchcompound upon the tissue sample is observed.

Various delivery systems are known and can be used to administer anantibody of the disclosure or a polynucleotide encoding an antibody ofthe disclosure, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the compound,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), construction of a nucleic acid as part of aretroviral or other vector, etc.

In a further embodiment, the compositions of the disclosure areadministered in combination with an antineoplastic agent, an antiviralagent, antibacterial or antibiotic agent or antifungal agents. Any ofthese agents known in the art may be administered in the compositions ofthe current disclosure.

In another embodiment, compositions of the disclosure are administeredin combination with a chemotherapeutic agent. Chemotherapeutic agentsthat may be administered with the compositions of the disclosureinclude, but are not limited to, antibiotic derivatives (e.g.,doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens(e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU,methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

Combination Compositions and Therapies

The anti-GM-CSF antibodies of the present disclosure can be used, insome embodiments, together with another therapeutic agent.

In some embodiment, the second therapeutic agent is an anti-inflammatoryagent. Non-limiting examples include aspirin, ibuprofen, naproxen,celecoxib (Celebrex), piroxicam (Feldene), indomethacin (Indocin),meloxicam (Mobic Vivlodex), ketoprofen (Orudis, Ketoprofen ER, Oruvail,Actron), sulindac (Clinoril), diflunisal (Dolobid), nabumetone(Relafen), oxaprozin (Daypro), tolmetin (Tolmetin Sodium, Tolectin),salsalate (Disalcid), etodolac (Lodine), fenoprofen (Nalfon),flurbiprofen (Ansaid), ketorolac (Toradol), meclofenamate, and mefenamicacid (Ponstel).

In some embodiments, the second therapeutic agent is suitable fortreating an autoimmune disease. Non-limiting examples includeglucocorticoid, an anti-CD3 antibodies such as Muromonab-CD3, IL-2ainhibitors such as basiliximab (Simulect) and daclizumab (Zenapax),calcineurin inhibitors such as tacrolimus and ciclosporin, sirolimus,everolimus, interferons, opioids, TNF-binding proteins or antibodies,and mycophenolate.

In some embodiment, the second therapeutic agent is a cancerchemotherapeutic agent. Chemotherapeutic agents may be categorized bytheir mechanism of action into, for example, the following groups:

-   -   anti-metabolites/anti-cancer agents such as pyrimidine analogs        floxuridine, capecitabine, and cytarabine;    -   purine analogs, folate antagonists, and related inhibitors;    -   antiproliferative/antimitotic agents including natural products        such as vinca alkaloid (vinblastine, vincristine) and        microtubule such as taxane (paclitaxel, docetaxel), vinblastin,        nocodazole, epothilones, vinorelbine (NAVELBINE®), and        epipodophyllotoxins (etoposide, teniposide);    -   DNA damaging agents such as actinomycin, amsacrine, busulfan,        carboplatin, chlorambucil, cisplatin, cyclophosphamide        (CYTOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin,        iphosphamide, melphalan, merchlorethamine, mitomycin,        mitoxantrone, nitrosourea, procarbazine, taxol, taxotere,        teniposide, etoposide, and triethylenethiophosphoramide;    -   antibiotics such as dactinomycin, daunorubicin, doxorubicin,        idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin        (mithramycin), and mitomycin;    -   enzymes such as L-asparaginase which systemically metabolizes        L-asparagine and deprives cells which do not have the capacity        to synthesize their own asparagine;    -   antiplatelet agents;    -   antiproliferative/antimitotic alkylating agents such as nitrogen        mustards cyclophosphamide and analogs (melphalan, chlorambucil,        hexamethylmelamine, and thiotepa), alkyl nitrosoureas        (carmustine) and analogs, streptozocin, and triazenes        (dacarbazine);    -   antiproliferative/antimitotic antimetabolites such as folic acid        analogs (methotrexate);    -   platinum coordination complexes (cisplatin, oxiloplatinim, and        carboplatin), procarbazine, hydroxyurea, mitotane, and        aminoglutethimide;    -   hormones, hormone analogs (estrogen, tamoxifen, goserelin,        bicalutamide, and nilutamide), and aromatase inhibitors        (letrozole and anastrozole);    -   anticoagulants such as heparin, synthetic heparin salts, and        other inhibitors of thrombin;    -   fibrinolytic agents such as tissue plasminogen activator,        streptokinase, urokinase, aspirin, dipyridamole, ticlopidine,        and clopidogrel;    -   antimigratory agents;    -   antisecretory agents (breveldin);    -   immunosuppressives tacrolimus, sirolimus, azathioprine, and        mycophenolate;    -   compounds (TNP-470, genistein) and growth factor inhibitors        (vascular endothelial growth factor inhibitors and fibroblast        growth factor inhibitors);    -   angiotensin receptor blockers, nitric oxide donors;    -   anti-sense oligonucleotides;    -   antibodies such as trastuzumab and rituximab;    -   cell cycle inhibitors and differentiation inducers such as        tretinoin;    -   inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin,        dactinomycin, eniposide, epirubicin, etoposide, idarubicin,        irinotecan, mitoxantrone, topotecan, and irinotecan), and        corticosteroids (cortisone, dexamethasone, hydrocortisone,        methylprednisolone, prednisone, and prednisolone);    -   growth factor signal transduction kinase inhibitors;    -   dysfunction inducers;    -   toxins such as Cholera toxin, ricin, Pseudomonas exotoxin,        Bordetella pertussis adenylate cyclase toxin, diphtheria toxin,        and caspase activators;    -   and chromatin.

Compositions

The present disclosure also provides pharmaceutical compositions. Suchcompositions comprise an effective amount of an antibody, and anacceptable carrier. In some embodiments, the composition furtherincludes a second therapeutic agent.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Further, a “pharmaceutically acceptable carrier” will generally be anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents such as acetates,citrates or phosphates. Antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences by E. W. Martin, incorporated herein byreference. Such compositions will contain a therapeutically effectiveamount of the antigen-binding polypeptide, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

EXAMPLES Example 1. Generation of Murine Antibodies

This example describes the process of preparing anti-human-GM-CSF mousemonoclonal antibodies using the hybridoma technology. Recombinant humanGM-CSF protein was used as antigen. To generate mouse monoclonalantibodies to human GM-CSF, different strains of 6-8 week mice includingBALB/c, C57/BL6 or SJL mice were firstly immunized with 20 μgrecombinant human GM-CSF. On day 14, 28 and 42 post-first immunization,the immunized mice were re-immunized with 5 μg recombinant protein. Toselect mice producing antibodies that bond the GM-CSF protein, sera fromimmunized mice were tested by ELISA. Briefly, microtiter plates werecoated with human GM-CSF protein at 1 μg/ml in PBS, 100 μl/well at roomtemperature (RT) overnight, then blocked with 100 μl/well of 5% BSA.Dilutions of plasma from immunized mice were added to each well andincubated for 1-2 hours at RT. The plates were washed with PBS/Tween andthen incubate with anti-mouse IgG antibody conjugated with Horse RadishPeroxidase (HRP) for 1 hour at RT. After washing, the plates weredeveloped with ABTS substrate and analyzed by spectrophotometer at OD405 nm. Mice with sufficient titers of anti-GM-CSF IgG were boosted with25 μg recombinant human GM-CSF protein at Day 60 post-immunization. Theresulting mice were used for fusions.

The hybridoma supernatants were tested for anti-GM-CSF IgGs by ELISAscreening. The primary ELISA positive hybridoma clones were selected forsubcloning using limited dilution method and further tested byconfirmatory ELISA binding assay (FIG. 1) and TF-1 proliferation assay(FIG. 2). Prior to GM-CSF stimulation, TF-1 cells were washed withRPMI1640 basal medium and starved for over-night. At day 2, thesestarved cells were collected and then seeded at a concentration of 3×10⁵cells/ml in 50 μl per well of a flat bottom 96 well cell culture plate.

Human recombinant GM-CSF (Genscript) at a concentration of 0.2 ng/ml(4×) was 1:1 mixed 20% hybridoma culture supernatant (4×) and 50 μl ofthe mix was added to the TF-1 cells, so the final concentration forGM-CSF was 0.05 ng/ml and for hybridoma supernatant was 5%. Maximal cellproliferation (0% inhibition) was measured incubating TF-1 cells at afinal concentration of GM-CSF of 0.05 ng/ml, without the addition ofhybridoma supernatant. 100% inhibition of TF-1 proliferation wasmeasured by omitting GM-CSF from the assay and keeping the cells inRPMI1640 complete medium only. TF-1 cells were then incubated for 72 hrsat 37° C. Cell viability was measured by CellTiter-Glo® Luminescent CellViability Assay according to the manufacturer's protocol.

Overall there were three hybridoma monoclones 23F4, 32C4 and 5005 thatshowed significant inhibition of TF-1 proliferation while 23F4 and 50C5were selected for further characterization.

Example 2. Binding of the Murine Antibodies to Human or Rhesus GM-CSF

This example tests the dose response of ELISA binding of mouseanti-GM-CSF mAb to recombinant human or rhesus GM-CSF protein (1μg/ml@100 μl).

Recombinant human or rhesus GM-CSF protein (Genscript) was coated at 1μg/ml in PBS onto microtiter plates for 2 h at room temperature (RT).After coating of antigen the wells were blocked with PBS/0.05% Tween(PBST) with 1% BSA for 1 h at RT. After washing of the wells with PBST,different concentrations of anti-GM-CSF antibodies were added to thewell and incubated for 1 at RT. For detection of the binding antibodies,the HRP-conjugated secondary antibodies against mouse Fc (Jackson ImmunoResearch) were added, followed by the addition of fluorogenic substrates(Roche). Between all incubation steps, the wells of the plate werewashed with PBST three times. Fluorescence was measured in a TECANSpectrafluor plate reader.

As shown in FIG. 3, both 23F4 and 50C5 antibodies showed dose-dependentbinding to human GM-CSF with EC50s of 11.8 ng/ml and 14.6 ng/ml,respectively, and rhesus GM-CSF with EC50s of 10.2 ng/ml and 21.7 ng/ml,respectively.

Example 3. Binding Kinetics of the Murine Antibodies to Human GM-CSF

FIG. 4 plots the binding kinetics of 23F4 and 50C5 with recombinanthuman GM-CSF. Recombinant human GM-CSF was set as an analyte with serialconcentrations (100, 50, 25, 12.5, 6.25, 3.125 nM). The binding kineticsassay of antibody to antigen was performed using Biacore T200 systemthrough a mouse antibody capture approach. The anti-mouse Fc IgG wereimmobilized on CMS sensor chip according to the manufacturer'sinstruction. The test antibody was injected and captured by theimmobilized anti-mouse Fc IgG. And then serial concentrations of theantigen were individually injected, and the binding profile was recordedfor each concentration of antigen analyte, respectively. The assaysystem was regenerated by injection of 10 mM Glycine-HCL pH 1.5 for 30seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mMNaCl, 3 mM EDTA and 0.05% P20). The assay temperature was 25° C., andthe association and dissociation time were 180 and 600 seconds,respectively.

The Biacore data were fitted using Biacore T200 evaluation software 1.0according to 1:1 binding model to calculate the association (ka) anddissociation (kd) rate constants as well as the equilibrium constant(KD). In addition to FIG. 4, some summary data presented in the tablebelow.

Sample ka (1/Ms) kd (1/s) KD (M) 23F4 8.723E+05 3.635E−05 4.168E−11 50C52.718E+06 1.204E−04 4.431E−11

Example 4. Blocking of GM-CSF Binding to the GM-CSF Receptor Alpha bythe Murine Antibodies

In order to test the potency of the antibodies in the blockade of GM-CSFbinding to the GM-CSF receptor alpha chain, recombinant human GM-CSFreceptor alpha protein (CD116) was coated at 2 μg/ml in PBS ontomicrotiter plates for over-night at 4° C. After coating of antigen thewells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h atRT. After washing of the wells with PBST, different concentrations ofanti-GM-CSF antibodies were added to the well in the presence ofbiotinylated human GM-CSF protein (0.05 μg/ml) and incubated for 1 hr atRT.

For detection of the binding of biotinylated GM-CSF to the coatedreceptor, the HRP-conjugated Streptavidin was added, followed by theaddition of fluorogenic substrates (Roche). Between all incubationsteps, the wells of the plate were washed with PBST three times.Fluorescence was measured in a TECAN Spectrafluor plate reader. As shownin FIG. 5, both antibodies showed dose-dependent inhibition of GM-CSFbinding to the GM-CSF receptor alpha.

Example 5. Blocking of GM-CSF by the Murine Antibodies Induced pSTAT5Signaling

CD14+monocytes were purified from peripheral human blood by using CD14positive microbeads (Miltenyi Biotec). The purified monocytes werestimulated with human GM-CSF (0.2 ng/ml) for 30 minutes at 37° C. in thepresence of different concentrations of 23F4 antibody. After incubation,the cells were collected and washed with FACS buffer (1× PBS+2% FBS) andpermealized by 2% PFA followed by cell fixation using ice cold methanol.Then the PE-conjugated anti-pSTAT5 antibody was added to the cells foranother incubation of 30 minutes at 4° C. and analyzed by flowcytometry. % of inhibition was calculated by [1−(MFI test sample/MFIcontrol)]×100%. Addition of the antibodies could significantly decreasethe level of pSTAT5 activation induced by GM-CSF at a dose of 0.1 or 1μg/ml (FIG. 6).

Example 6. Inhibition of GM-CSF Dependent TF-1 Proliferation by theMurine Antibodies

Prior to GM-CSF stimulation, TF-1 cells were washed with RPMI1640 basalmedium and starved for over-night. At day 2, these starved cells werecollected and then seeded at a concentration of 3×105 cells/ml in 50 μlper well of a flat bottom 96 well cell culture plate. Human recombinantGM-CSF (Genscript) at a concentration of 0.2 ng/ml (4×) was 1:1 mixedwith murine anti-GM-CSF antibodies (0.01 ng/ml-1000 ng/ml diluted incomplete medium) and 50 μl of the mix was added to the TF-1 cells.Maximal cell proliferation (0% inhibition) was measured incubating TF-1cells at a final concentration of GM-CSF of 0.05 ng/ml, without theaddition of antibody. 100% inhibition of TF-1 proliferation was measuredby omitting GM-CSF from the assay and keeping the cells in RPMI1640complete medium only. TF-1 cells were then incubated for 72 hrs at 37°C. Cell viability was measured by CellTiter-Glo® Luminescent CellViability Assay according to the manufacturer's protocol. The IC50 valuefor 23F4 and 50C5 in the inhibition of TF-1 proliferation were bothabout 10.9 ng/ml (FIG. 7).

Example 7. Humanization of the Murine Antibodies

The murine antibodies' variable region genes were employed to createhumanized MAb. In the first step of this process, the amino acidsequences of the VH and VK of the antibodies were compared against theavailable database of human Ig gene sequences to find the overallbest-matching human germline Ig gene sequences.

Humanized variable domain sequences were then designed where the CDR1, 2and 3 of the antibody heavy and light chains were grafted onto frameworksequences of the human Ig genes. A 3D model was then generated todetermine if there were any framework positions where replacing themouse amino acid to the human amino acid (back mutations) could affectbinding and/or CDR conformation. The relevant sequences and backmutations are shown in the tables below.

CDR Sequences of 23F4 VH

CDR1 (SEQ ID NO. 1) SHYLH CDR2 (SEQ ID NO. 2) WIFPGDDKTKYNEKFKG CDR3(SEQ ID NO. 3) GTKYLNWNFDV

CDR Sequences of 23F4 VL

CDR1 (SEQ ID NO. 4) KANQNVGTTLA CDR2 (SEQ ID NO. 5) SASYRYS CDR3(SEQ ID NO. 6) HQYTTYPLT

Humanization Design for 23F4

Construct Mutation VH Design I: VH1-f/JH6 23F4 VH Chimera 23F4 VH.1CDR-grafted, Q1E, T98R 23F4 VH.1a Based on 23F4 VH.1, A72S 23F4 VH.1bBased on 23F4VH.1, A72S, V68A 23F4 VH.1c Based on 23F4VH.1, A72S, V68A,I70L, M48I 23F4 VH.1d Based on 23F4VH.1, A72S, V68A, I70L, M48I, G26D,F29L VH Design II: VH1-f/JH6 23F4 VH.2 CDR-grafted, Q1E 23F4 VH.2a Basedon 23F4 VH.2, R72S 23F4 VH.2b Based on 23F4 VH.2, R72S, M70L 23F4 VH.2cBased on 23F4 VH.2, R72S, M70L, M48I, V68A 23F4 VH.2d Based on 23F4VH.2, R72S, M70L, M48I, V68A, G26D, F29L VK Design: L8/Jk4 23F4VkChimera 23F4 Vk.1 CDR-grafted 23F4 Vk.1a Based on 23F4 Vk.1, L46A 23F4Vk.1b Based on 23F4 Vk.1, L46A, S60D, E70D 23F4 Vk.1c Based on 23F4Vk.1, L46A, S60D, E70D, A43S, Y87F

The amino acid and nucleotide sequences of some of the humanizedantibody 23F4 are listed in the table below.

Humanized antibody sequences (CDR residues are underlined and backmutations are in Indicated Boxes)

23F4 VH QVQLQQSGPELVKPGTSMKISCKTSDYTLT SHYLH WVKQRPGQGLEWIG WSEQ ID NO. 7 IFPGDDKTKYNEKFKG KATLTSDKTSNTAYMQLSSLTSEESAVYFCAR GTKYLNWNFDV WGTGTTVTVSS 23F4 VH.1

SEQ ID NO. 8

KYLNWNFDV WGQGTTVTVSS 23F4 VH.1a

SEQ ID NO. 9

KYLNWNFDV WGQGTTVTVSS 23F4 VH.1b

SEQ ID NO. 10

KYLNWNFDV WGQGTTVTVSS 23F4 VH.1c

SEQ ID NO. 11

KYLNWNFDV WGQGTTVTVSS 23F4 VH.1d

SEQ ID NO. 12

KYLNWNFDV WGQGTTVTVSS 23F4 VH.2

SEQ ID NO. 13

KYLNWNFDV WGQGTTVTVSS 23F4 VH.2a

SEQ ID NO. 14

KYLNWNFDV WGQGTTVTVSS 23F4 VH.2b

SEQ ID NO. 15

KYLNWNFDV WGQGTTVTVSS 23F4 VH.2c

SEQ ID NO. 16

KYLNWNFDV WGQGTTVTVSS 23F4 VH.2d

SEQ ID NO. 17

KYLNWNFDV WGQGTTVTVSS 23F4Vk DIVLTQPQKFLSTSVGDRVSVTC KANQNVGTTLAWYQQKPGQSPKALIY S SEQ ID NO. 18 ASYRYS GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYTTYPLT FGG GTKLEIK 23F4 Vk.1 DIQLTQSPSFLSASVGDRVTITC KANQNVGTTLAWYQQKPGKAPKLLIY S SEQ ID NO. 19 ASYRYS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQYTTYPLT FGG GTKVEIK 23F4 Vk.1a

SEQ ID NO. 20 ASYRYS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC HQYTTYPLT FGGGTKVEIK 23F4 Vk.1b

SEQ ID NO. 21

GTKVEIK 23F4 Vk.1c

SEQ ID NO. 22

GTKVEIK

Combination of VH/VK for Humanization Antibody 23F4

Vk VH 23F4 Vk.1 23F4 Vk.1a 23F4 Vk.1b 23F4 Vk.1c 23F4 Vk 23F4 VH.1Hu23F4-1 Hu23F4-2 Hu23F4-3 Hu23F4-4 23F4 VH.1a Hu23F4-5 Hu23F4-6Hu23F4-7 Hu23F4-8 23F4 VH.1b Hu23F4-9 Hu23F4-10 Hu23F4-11 Hu23F4-12 23F4VH.1c Hu23F4-13 Hu23F4-14 Hu23F4-15 Hu23F4-16 23F4 VH.1d Hu23F4-17Hu23F4-18 Hu23F4-19 23F4 VH.2 Hu23F4-20 Hu23F4-21 Hu23F4-22 Hu23F4-2323F4 VH.2a Hu23F4-24 Hu23F4-25 Hu23F4-26 Hu23F4-27 23F4 VH.2b Hu23F4-28Hu23F4-29 Hu23F4-30 Hu23F4-31 23F4 VH.2c Hu23F4-32 Hu23F4-33 Hu23F4-34Hu23F4-35 23F4 VH.2d Hu23F4-36 Hu23F4-37 Hu23F4-38 23F4 VH 23F4 chimera

CDR Sequences of 5005 VH

CDR1 (SEQ ID NO. 23) PYSIH CDR2 (SEQ ID NO. 24) YINPSTGYIEYNQHFKD CDR3(SEQ ID NO. 25) GGDYEGYFDY

CDR Sequences of 5005 VL

CDR1 (SEQ ID NO. 26) RLNENIYSFLA CDR2 (SEQ ID NO. 27) NAETLAE CDR3(SEQ ID NO. 28) QQHYGTPYT

Humanization Design for 5005

Construct Mutation VH Design I: VH1-69/JH6 50C5 VH Chimera 50C5 VH.1CDR-grafted, Q1E 50C5 VH.1a Based on 50C5 VH.1, S84R 50C5 VH.1b Based on50C5 VH.1, S84R, G27Y, T28I 50C5 VH.1c Based on 50C5 VH.1, S84R, G27Y,T28I, M48I 50C5 VH.1d Based on 50C5 VH.1, S84R, G27Y, T28I, M48I, V68T,I70L 50C5 VH.1e Based on 50C5 VH.1, S84R, G27Y, T28I, M48I, V68T, I70L,S30T VK Design: O12/Jk4 50C5Vk Chimera 50C5 Vk.1 CDR-grafted 50C5 Vk.1aBased on 50C5 Vk.1, 148V 50C5 Vk.1b Based on 50C5 Vk.1, 148V, G57D 50C5Vk.1c Based on 50C5 Vk.1, 148V, G57D, D70Q 50C5 Vk.1d Based on 50C5Vk.1, 148V, G57D, D70Q, A43S

Humanized Antibody Sequences (CDR Residues are Underlined and BackMutations are in Indicated Boxes)

50C5 VH QVQLQQSAAELVRPGASVKMSCKASGYIFT PYSIH WIKQRPGQGLEWIG YSEQ ID NO. 29 INPSTGYIEYNQHFKD RTTLTADKSSSTAYMQLRSLTSEDSAVYYCAR GGDYEGYFDY WGQGTTLTVSS 50C5 VH.1

SEQ ID NO. 30 INPSTGYIEYNQHFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GGDYEGYFDY WGQGTTVTVSS 50C5 VH.1a

SEQ ID NO. 31

DYEGYFDY WGQGTTVTVSS 50C5 VH.1b

SEQ ID NO. 32

DYEGYFDY WGQGTTVTVSS 50C5 VH.1c

SEQ ID NO. 33

DYEGYFDY WGQGTTVTVSS 50C5 VH.1d

SEQ ID NO. 34

DYEGYFDY WGQGTTVTVSS 50C5 VH.1e

SEQ ID NO. 35

DYEGYFDY WGQGTTVTVSS 50C5 Vk DIQMTQSPDSLSASVGETVTITC RLNENIYSFLAWYQQRQGKSPQLLVY NA SEQ ID NO. 36 ETLAE DVPSRFSGSGSGTQFSLKISSLQTDDFGTYYCQQHYGTPYT FGGGT NLEIE 50C5 Vk.1 DIQMTQSPSSLSASVGDRVTITC RLNENIYSFLAWYQQKPGKAPKLLIY NA SEQ ID NO. 37 ETLAE GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYGTPYT FGGGT KVEIK 50C5 Vk.1a

SEQ ID NO. 38 ETLAE GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYGTPYT FGGGTKVEIK 50C5 Vk.1b

SEQ ID NO. 39

KVEIK 50C5 Vk.1c

SEQ ID NO. 40

KVEIK 50C5 Vk.1d

SEQ ID NO. 41

KVEIK

Combination of VH/VK for Humanization Antibody 5005

Vk VH 50C5 Vk.1 50C5 Vk.1a 50C5 Vk.1b 50C5 Vk.1c 50C5 Vk.1d 50C5 Vk 50C5VH.1 50C5 VH.1a Hu50C5-1 Hu50C5-2 Hu50C5-3 Hu50C5-4 Hu50C5-5 50C5 VH.1bHu50C5-6 Hu50C5-7 Hu50C5-8 Hu50C5-9 Hu50C5-10 50C5 VH.1c Hu50C5-11Hu50C5-12 Hu50C5-13 Hu50C5-14 Hu50C5-15 50C5 VH.1d Hu50C5-16 Hu50C5-17Hu50C5-18 Hu50C5-19 Hu50C5-20 50C5 VH.1e Hu50C5-21 Hu50C5-22 Hu50C5-23Hu50C5-24 50C5 VH 50C5 chimera

Example 8. Binding of Humanized Antibodies to Human GM-CSF

The humanized variants were tested for the binding to recombinant humanGM-CSF as previously described. Recombinant human GM-CSF protein(Genscript) was coated at 1 ug/ml in PBS onto microtiter plates for 2 hat room temperature (RT). After coating of antigen the wells wereblocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at RT. Afterwashing of the wells with PBST, different concentrations of anti-GM-CSFhumanized antibodies were added to the well and incubated for 1 at RT.

For detection of the binding antibodies, the HRP-conjugated secondaryantibodies against mouse Fc (Jackson Immuno Research) were added,followed by the addition of fluorogenic substrates (Roche). Between allincubation steps, the wells of the plate were washed with PBST threetimes. Fluorescence was measured in a TECAN Spectrafluor plate reader.As shown in FIG. 8, all the humanized variants demonstrated a similarbinding potency against human GM-CSF as compared with chimeric antibody.

Example 9. Binding Kinetics of Humanized Antibodies

The binding kinetics of humanized antibodies were measured by Biacore aspreviously described. Recombinant human GM-CSF was set as an analytewith serial concentrations (100, 50, 25, 12.5, 6.25, 3.125 nM). Thebinding kinetics assay of antibody to antigen was performed usingBiacore T200 system through a human antibody capture approach. Theanti-human Fc IgG were immobilized on CMS sensor chip according to themanufacturer's instruction. The test antibody was injected and capturedby the immobilized anti-human Fc IgG. And then serial concentrations ofthe antigen were individually injected, and the binding profile wasrecorded for each concentration of antigen analyte, respectively.

The assay system was regenerated by injection of 10 mM Glycine-HCL pH1.5 for 30 seconds. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4,150 mM NaCl, 3 mM EDTA and 0.05% P20). The assay temperature was 25° C.,and the association and dissociation time were 180 and 600 seconds,respectively. The Biacore data were fitted using Biacore T200 evaluationsoftware 1.0 according to 1:1 binding model to calculate the association(ka) and dissociation (kd) rate constants as well as the equilibriumconstant (1(D). As shown in the tables below, from 23F4, Hu23F4-13,Hu23F4-27 and Hu23F4-36 demonstrated the strongest binding affinity ascompared with chimeric antibody; from 5005, Hu50C5-8, Hu50C5-17,Hu50C5-18, Hu50C5-19, Hu50C5-21 and Hu50C5-23 demonstrated the strongestbinding affinity as compared with chimeric antibody.

Antigen Antibody ka (1/Ms) kd (1/s) KD (M) GM-CSF 3523-Hu23F4-5 1.188E+61.239E−4 1.043E−10 3523-Hu23F4-6 8.76SE+S 1.353E−4 1.544E−103523-Hu23F4-13 1.279E+6 1.195E−4 9.349E−11 3523-Hu23F4-20 8.007E+51.137E−4 1.420E−10 3523-Hu23 F4-23 8.994E+5 1.164E−4 1.295E−10 3523-Hu23F4-25 6.354E+5 1.091E−4 1.718E−10 3523-Hu23F4-27 9.232E+5 9.461E−51.025E−10 3523-Hu23F4-29 6.673E+S 1.013E−4 1.518E−10 3523-Hu23F4-361.271E+6 1.026E−4 8.070E−11 3523-Hu23F4- 1.229E+6 3.873E−5 3.150E−11chimera 35230Hu50C5-6 4.418E+6 1.375E−4 3.112E−11 35230Hu50C5-8 4.911E+61.386E−4 2.822E−11 35230Hu50C5-11 4.368E+6 1.363E−4 3.121E−1135230Hu50C5-17 4.466E+6 1.112E−4 2.491E−11 35230Hu50C5-19 4.724E+61.089E−4 2.306E−11 35230Hu50C5-19 4.622E+6 1.061E−4 2.295E−1135230Hu50C5-21 4.592E+6 1.082E−4 2.356E−11 35230Hu50C5-23 4.535E+61.251E−4 2.760E−11 35230Hu50C5- 4.433E+6 1.419E−4 3.202E−11 chimera

Example 10. Inhibition of TF-1 Proliferation by Humanized Antibodies

Prior to GM-CSF stimulation, TF-1 cells were washed with RPMI1640 basalmedium and starved for over-night. At day 2, these starved cells werecollected and then seeded at a concentration of 3×105 cells/ml in 50 ulper well of a flat bottom 96 well cell culture plate. Human recombinantGM-CSF (Genscript) at a concentration of 0.2 ng/ml (4×) was 1:1 mixedwith humanized anti-GM-CSF antibodies (0.01 ng/ml-1000 ng/ml diluted incomplete medium) and 50 ul of the mix was added to the TF-1 cells.Maximal cell proliferation (0% inhibition) was measured incubating TF-1cells at a final concentration of GM-CSF of 0.05 ng/ml, without theaddition of antibody. 100% inhibition of TF-1 proliferation was measuredby omitting GM-CSF from the assay and keeping the cells in RPMI1640complete medium only. TF-1 cells were then incubated for 72 hrs at 37°C. Cell viability was measured by CellTiter-Glo® Luminescent CellViability Assay according to the manufacturer's protocol. Among thehumanized antibodies from 23F4 tested, Hu23F4-13, Hu23F4-27 andHu23F4-36 showed strongest inhibition with an IC50 of 4.95 ng/ml, 3.95ng/ml and 3.30 ng/ml, respectively (FIG. 9). Among the humanizedantibodies from 5005 tested, Hu50C5-23 showed strongest inhibition withan IC50 of 14.31 ng/ml (FIG. 9).

Antibody name IC50 for TF-1 proliferation Hu23F4-chimera 8.55 ng/mlHu23F4-5 15.58 ng/ml Hu23F4-6 7.87 ng/ml Hu23F4-13 4.95 ng/ml Hu23F4-208.77 ng/ml Hu23F4-23 9.08 ng/ml Hu23F4-25 12.22 ng/ml Hu23F4-27 3.95ng/ml Hu23F4-29 27.44 ng/ml Hu23F4-36 3.30 ng/ml Hu50C5-chimera 86.47ng/ml Hu50C5-6 23.93 ng/ml Hu50C5-8 45.90 ng/ml Hu50C5-11 111.1 ng/mlHu50C5-17 22.06 ng/ml Hu50C5-18 41.27 ng/ml Hu50C5-19 16.99 ng/mlHu50C5-21 21.64 ng/ml Hu50C5-23 14.31 ng/ml

Example 11. Blocking of pSTAT5 Signaling By Humanized Antibodies

CD14+monocytes were purified from peripheral human blood by using CD14positive microbeads (Miltenyi Biotec). The purified monocytes werestimulated with human GM-CSF (0.2 ng/ml) for 30 minutes at 37° C. in thepresence of different concentrations of humanized antibodies. Afterincubation, the cells were collected and washed with FACS buffer(1×PBS+2% FBS) and permealized by 2% PFA followed by cell fixation usingice cold methanol. Then the PE-conjugated anti-pSTAT5 antibody was addedto the cells for another incubation of 30 minutes at 4° C. and analyzedby flow cytometry. % of inhibition was calculated by [1−(MFI testsample/MFI control)]×100%. Addition of Hu23F4-13, Hu23F4-27, Hu23F4-36,Hu50C5-17 and Hu50C5-23 could significantly decrease the level of pSTAT5activation induced by GM-CSF at a dose of 0.1 or 1 μg/ml (FIG. 10).

Example 12. Binding of Humanized Antibodies to Rhesus GM-CSF

Recombinant rhesus GM-CSF protein (Genscript) was coated at 1 ug/ml inPBS onto microtiter plates for 2 h at room temperature (RT). Aftercoating of antigen, the wells were blocked with PBS/0.05% Tween (PBST)with 1% BSA for 1 h at RT. After washing of the wells with PBST,different concentrations of humanized anti-GM-CSF antibodies were addedto the well and incubated for 1 at RT. For detection of the bindingantibodies, the HRP-conjugated secondary antibodies against mouse Fc(Jackson Immuno Research) were added, followed by the addition offluorogenic substrates (Roche). Between all incubation steps, the wellsof the plate were washed with PBST three times. Fluorescence wasmeasured in a TECAN Spectrafluor plate reader. As shown in FIG. 11,Hu23F4-13, Hu23F4-27 and Hu23F4-36 showed a dose-dependent binding torhesus GM-CSF with an EC50 of 7.44 ng/ml, 6.25 ng/ml and 7.75 ng/ml,respectively; Hu50C5-17 and Hu50C5-23 showed a dose-dependent binding torhesus GM-CSF with an EC50 of 18.86 ng/ml and 21.63 ng/ml, respectively.

Antibody name EC50 for rhesus GM-CSF binding Hu23F4-13 7.44 ng/mlHu23F4-27 6.25 ng/ml Hu23F4-36 7.75 ng/ml Hu50C5-17 18.86 ng/mlHu50C5-23 21.63 ng/ml

Example 13. Pharmacokinetics of Hu23F4-27 in Cynomolgus Monkey

From the previous results of in vitro bio-activity assays, Hu23F4-27 wasselected to determine the pharmacokinetics properties in naïvecynomolgus monkey. Hu23F4-27 antibody was administered by bolusintravenous injection to naïve cynomolgus monkey at different doses of0.4 mg/kg, 2 mg/kg and 10 mg/kg, respectively. Plasma samples werecollected at selected timepoints out to 28 days after dosing, and theconcentration of the respective protein determined by ELISA. Thepharmacokinetics parameters were then calculated using anon-compartmental approach with WinNonlin (Certara, CA) and shown inFIG. 12.

Group T_(1/2) C_(max) AUC_(0-t) AUC_(inf) CL (n = 2) (h) (μg/ml)(Day*μg/ml) (day*μg/ml) (ml/hr/kg) 10 mg/kg 121.4 ± 11.4 432.2 ± 31.71254.5 ± 38.3  1276.1 ± 48.6  0.327 ± 0.012 2 mg/kg 220.7 ± 94.8 74.1 ±8.3 280.3 ± 46.3 311.4 ± 76.6 0.276 ± 0.068 0.4 mg/kg 178.2 ± 4.8  11.1± 4.6 49.4 ± 1.9 52.9 ± 1.7 0.315 ± 0.010

The present disclosure is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the disclosure, and any compositions or methodswhich are functionally equivalent are within the scope of thisdisclosure. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present disclosure without departing from the spirit or scope ofthe disclosure. Thus, it is intended that the present disclosure coverthe modifications and variations of this disclosure provided they comewithin the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. An isolated antibody or fragment thereof, wherein the antibody orfragment thereof has specificity to a human GM-CSF protein and comprisesa VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VLCDR3 of SEQ ID NO:
 6. 2. The antibody or fragment thereof of claim 1,further comprising a heavy chain constant region, a light chain constantregion, an Fc region, or the combination thereof.
 3. The antibody orfragment thereof of claim 1, wherein the light chain constant region isa kappa or lambda chain constant region.
 4. The antibody or fragmentthereof of claim 1, wherein the antibody or fragment thereof is of anisotype of IgG, IgM, IgA, IgE or IgD.
 5. (canceled)
 6. (canceled)
 7. Theantibody or fragment thereof of claim 6, wherein the antibody orfragment thereof is a humanized antibody.
 8. The antibody or fragmentthereof of claim 7, comprising a heavy chain variable region comprisingone or more amino acid residues selected from the group consisting of:(a) Glu at position 1, (b) Arg at position 98, (c) Ser at position 72,(d) Ala at position 68, (e) Leu at position 70, (f) Ile at position 48,(g) Asp at position 26, and (h) Leu at position 29, according to Kabatnumbering, and combinations thereof.
 9. The antibody or fragment thereofof claim 8, wherein the heavy chain variable region comprises at least(a) Glu at position
 1. 10. The antibody or fragment thereof of claim 8,wherein the heavy chain variable region comprises a fragment of DYTLT(SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) starting at position 26according to Kabat numbering.
 11. The antibody or fragment thereof ofclaim 7, comprising a light chain variable region comprising one or moreamino acid residues selected from the group consisting of: (a) Ala atposition 46, (b) Asp at position 60, (c) Asp at position 70, (d) Ser atposition 43, and (f) Phe at position 87, according to Kabat numbering,and combinations thereof.
 12. The antibody or fragment thereof of claim1, comprising a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 8-17, or apeptide having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 8-17.
 13. (canceled)14. The antibody or fragment thereof of claim 1, comprising a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 19-22, or a peptide having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 19-22.
 15. (canceled)
 16. The antibody orfragment thereof of claim 1, wherein the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 14 and the light chainvariable region comprises the amino acid sequence of SEQ ID NO:
 22. 17.(canceled)
 18. A composition comprising the antibody or fragment thereofof claim 1 and a pharmaceutically acceptable carrier.
 19. (canceled) 20.A method of treating an inflammatory or autoimmune disease or conditionin a patient in need thereof, comprising administering to the patientthe antibody or fragment thereof of claim
 1. 21. The method of claim 20,wherein the inflammatory disease or condition is selected from the groupconsisting of Alzheimer's disease, Addison's disease, atherosclerosis,ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoidarthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis,asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD),Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia,hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous(SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerativecolitis.
 22. The method of claim 20, wherein the autoimmune disease orcondition is selected from the group consisting of alopecia areata,autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis,diabetes (type 1), celiac disease, autoimmune juvenile idiopathicarthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome,idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmunemyocarditis, multiple sclerosis, pemphigus/pemphigoid, perniciousanemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis,psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis,Sjögren's syndrome, systemic lupus erythematosus, autoimmunethyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, andgranulomatosis with polyangiitis (Wegener's).
 23. A method of treating acancer in a patient in need thereof, comprising administering to thepatient the antibody or fragment thereof of claim
 1. 24. The method ofclaim 23, wherein the cancer is selected from the group consisting ofbladder cancer, breast cancer, colorectal cancer, endometrial cancer,esophageal cancer, head and neck cancer, kidney cancer, leukemia, livercancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostatecancer, and thyroid cancer.
 25. A method of reducing or relieving painin a patient in need thereof, comprising administering to the patientthe antibody or fragment thereof of claim
 1. 26. (canceled)